Anti-Helicobacter pylori activity and Structure-Activity Relationship study of 2-Alkylthio-5-(nitroaryl)-1,3,4-thiadiazole Derivatives.

Nitro-containing heteroaromatic derivatives structurally related to nitroimidazole (Metronidazole) are being extensively evaluated against Helicobacter pylori isolates. On the other hand, 1,3,4-thiadiazole derivatives have also demonstrated promising antibacterial potential. In present study, we evaluated anti-H. pylori activity of novel hybrid molecules bearing nitroaryl and 1,3,4-thiadiazole moieties. Anti-H. pylori activity of novel 5-(5-nitroaryl)-1,3,4-thiadiazole derivatives bearing different bulky alkylthio side chains at C-2 position of thiadiazole ring, were assessed against three different metronidazole resistant H. pylori isolates by paper disk diffusion method. Most of the compounds demonstrated moderate to strong inhibitory response especially at 25 μg/disk. The structure-activity relationship study of the compounds demonstrated that introduction of different alkylthio moieties at C-2 position of thiadiazole ring; alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring. The promising compound of this scaffold, bearing 1-methyl-5-nitroimidazole moiety at C-5 and α-methylbenzylthio side chain at C-2 position of thiadiazole ring, showed strong inhibitory response against metronidazole resistant H. pylori isolates at 12.5 μg/disk (the inhibition zone diameter at all evaluated concentrations (12.5- 100 μg/disk) is >50 mm). Novel 5-(5-nitroaryl)-1,3,4-thiadiazole scaffold bearing different C-2 attached thio-pendant moieties with promising anti-H. pylori potential were identified. Among different nitroheterocycles, 5-nitrofuran and 5-nitroimidazole moieties were preferable for the substitution at C-5 position of 1,3,4-thiadiazole ring. Introduction of different alkylthio side chains at C-2 position of central ring alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring.

macromolecules. Therefore; the nitohetrocycle moiety is essential for the antimicrobial activity of these derivatives (18).
2-The 1,3,4-thiadiazole ring (B) could partly mimic the methyleneaminooxazolidinone fragment of furazolidone and enhances the antibacterial and particularly anti-H. pylori properties of scaffold.
3-Substituted-thio side chain (C) of these novel derivatives could serve as a new part against the resistant strains of H. pylori and affects the potency of these derivatives against the resistant isolates of this organism.
We have previously reported the synthetic pathway of designed compounds "1-13". The chemical structure of these compounds is demonstrated in Table 1. The structure of these compounds was confirmed by elemental analysis, 1 H NMR and IR spectra (26).

Introduction
Helicobacter pylori (H. pylori), an invading gram negative microaerophilic bacterium causes common chronic gastric infection known as the major cause of gastric and gastrodudenal ulcer disease (1-2). In addition, chronic infection of H. pylori is attributed to adenocarcinoma and low grade B-cell mucosaassociated lymphoid tissue lymphoma of stomach and therefore; it is the first bacterium known as definite carcinogen by the World Health Organization's International Agency for Research on Cancer (3-5). The treatment of H. pylori is complicated and includes combination of proton-pump inhibitor (PPI) agents with two or more different antibiotics such as amoxicillin, clarithromycin, metronidazole, tetracycline, and levofloxacin for at least 7 days (2, 6). Although eradication of infection results in ulcer healing and reduces the risk of gastric cancer (7), however; emerging resistance to current antibiotics is encountered as a major concern in the treatment of H. pylori infection (8). From this, different factors such as poor patient compliance, common side effects of combination therapy and also significant cost of multi-drug therapy, limits the eradication of infection and therefore reveals the urgent need to search novel candidates for the eradication or prevention of infection (7-8).
Recent studies reported some novel natural sources (9-13) and synthetic scaffolds (14-15) as the therapeutic agents with significant inhibitory activity against H. pylori isolates. Among the different chemical structures, nitrocontaining heteroaromatic derivatives (such as nitrofuran and nitrothiophen derivatives) that are structurally related to nitroimidazole family (Metronidazole) are being extensively evaluated against Helicobacter pylori isolates (16). In this regard, the current nitroheterocyclic antibiotics, tinidazole and furazolidone have demonstrated promising anti-H. pylori properties to be used in place of metronidazole (17). The results of different studies indicate that the possible mechanism of antimicrobial activity of 5-nitoheterocycles involves the reduction of the 5-nitro group with the formation of reactive intermediates that can interact to various cell According to our ongoing research program on 1,3,4-thiadiazole derivatives as potent antiparasitic scaffold (20-26), herein, we report the anti-H. pylori activity of 2-alkylthio-5-(nitroaryl)-1,3,4-thiadiazole derivatives.

Bacterial isolates and culture conditions
Clinical H. pylori isolates from gastric biopsy specimens were obtained from Shariati Hospital (Tehran, Iran). Primary isolation was performed on a selective blood agar base (Oxoid, Basingstoke, UK) supplemented with horse blood (5%, v/v) and 1 Selectatab tablet (500 mg; Mast Diagnostics, Merseyside, UK). Following primary selective isolation, H. pylori bacteria cells were identified according to colony morphology, Gram staining, microaerophilic growth at 37 °C, oxidase +, catalase +, urease +, nitrate -, H 2 S, and hippurate hydrolysis. Growth of H. pylori was maintained at 37 °C for 3-5 days in an atmosphere of 5% O 2 , 15% CO 2 , and 80% N 2 in an anaerobic chamber (Hirayama, Tokyo, Japan). To maintain a moist atmosphere, a moist paper towel was placed in the chamber. Bacterial strains were stored at -70 °C in brain heart infusion broth (BHIB) (Difco, East Molesey, UK) containing 10% (v/v) fetal calf serum (FCS) and 15% (v/v) glycerol. Frozen clinical isolates were thawed and inoculated on Mueller-Hinton agar (MHA) plates (Oxoid), supplemented with 10% horse blood and incubated under microaerophilic conditions. Given the importance of inoculum homogeneity, cellular viability was controlled microscopically by morphological observation with Gram staining. In order to control the proportions of coccoid cells in the cultures, cultures were always used after 48 h of incubation, when they generally did not present coccoid forms. Bacterial growth was taken from the plates and resuspended in sterile saline. The inoculum was prepared to contain 5 × 10 7 CFU/ mL by adjusting the turbidity of the suspension to match the McFarland No. 2 standard.

Bacterial growth inhibition assay (disk diffusion method)
Growth inhibition was performed by the filter paper disk diffusion method on selective Brucella agar with 7% defibrinated horse blood under microaerophilic conditions at 37 °C. The samples were evaluated for their anti-Helicobacter activity, dissolved in dimethylsulfoxide (DMSO). All compounds were assayed against metronidazole-resistant H. pylori strains at 3 concentrations (100, 50, and 25 and 12.5 μg/disk); the surfaces of the Brucella blood agar plates were inoculated with 100 μL of bacterial suspensions. Blank standard disks (6 mm in diameter) were deposited on the plates and impregnated with 10 μL of different dilutions of test compounds. Following incubation for 3-5 days at 37 °C, the inhibition zone around each disk (average diameter), if any, was recorded. The control disks received 10 μL of DMSO. All tests were performed in triplicate and the antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by the tested compounds.
The one-way analysis of variance (ANOVA) followed by Tukey multiple comparisons was used to analyze the data. A value of p < 0.05 was considered as the significance level between the groups.

Regio-isomeric chlorine substitution and
The best inhibitory response in 5-nitroimidazole series could also be achieved by the introduction of α-methylbenzylthio-side chain; compound 13 with inhibition zone diameter of > 50 at all the tested concentrations. While introduction of this side chain in 5-nitrothiophen derivatives, generates compound with weak anti-H. pylori activity at all concentrations (compound 10).
Among different nitroaryls, 5-nitrofuran and 5-nitroimidazole moieties are preferable for substitution at C-5 position of 1,3,4-thiadiazole ring. Introduction of different substituted-thio side chains at C-2 position of central ring alter the inhibitory activity which is mainly dependent on the type of C-5 attached nitrohetercyclic ring.

Acknowledgment
This work was supported by a grant from Research Council of Kerman University of Medical Sciences.  Table 1.